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• W3037807978 abstract "•One-photon calcium imaging of brain activity can suffer from neuropil crosstalk•Targeting GCaMPs to the cell body reduces neuropil crosstalk•One-photon imaging of somatic GCaMP reduces artifactual spikes and correlations•Somatic GCaMPs can be used in multiple species, such as mice and zebrafish Methods for one-photon fluorescent imaging of calcium dynamics can capture the activity of hundreds of neurons across large fields of view at a low equipment complexity and cost. In contrast to two-photon methods, however, one-photon methods suffer from higher levels of crosstalk from neuropil, resulting in a decreased signal-to-noise ratio and artifactual correlations of neural activity. We address this problem by engineering cell-body-targeted variants of the fluorescent calcium indicators GCaMP6f and GCaMP7f. We screened fusions of GCaMP to natural, as well as artificial, peptides and identified fusions that localized GCaMP to within 50 μm of the cell body of neurons in mice and larval zebrafish. One-photon imaging of soma-targeted GCaMP in dense neural circuits reported fewer artifactual spikes from neuropil, an increased signal-to-noise ratio, and decreased artifactual correlation across neurons. Thus, soma-targeting of fluorescent calcium indicators facilitates usage of simple, powerful, one-photon methods for imaging neural calcium dynamics. Methods for one-photon fluorescent imaging of calcium dynamics can capture the activity of hundreds of neurons across large fields of view at a low equipment complexity and cost. In contrast to two-photon methods, however, one-photon methods suffer from higher levels of crosstalk from neuropil, resulting in a decreased signal-to-noise ratio and artifactual correlations of neural activity. We address this problem by engineering cell-body-targeted variants of the fluorescent calcium indicators GCaMP6f and GCaMP7f. We screened fusions of GCaMP to natural, as well as artificial, peptides and identified fusions that localized GCaMP to within 50 μm of the cell body of neurons in mice and larval zebrafish. One-photon imaging of soma-targeted GCaMP in dense neural circuits reported fewer artifactual spikes from neuropil, an increased signal-to-noise ratio, and decreased artifactual correlation across neurons. Thus, soma-targeting of fluorescent calcium indicators facilitates usage of simple, powerful, one-photon methods for imaging neural calcium dynamics. Methods for one-photon fluorescent imaging of calcium dynamics are popular for neural activity mapping in the living brain. These techniques capture, at high speeds (e.g., >20 Hz), the dynamics of hundreds of neurons across large fields of view at a low equipment complexity and cost (Alivisatos et al., 2013Alivisatos A.P. Andrews A.M. Boyden E.S. Chun M. Church G.M. Deisseroth K. Donoghue J.P. Fraser S.E. Lippincott-Schwartz J. Looger L.L. et al.Nanotools for neuroscience and brain activity mapping.ACS Nano. 2013; 7: 1850-1866Crossref PubMed Scopus (245) Google Scholar, Grienberger and Konnerth, 2012Grienberger C. Konnerth A. Imaging calcium in neurons.Neuron. 2012; 73: 862-885Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar, Keller et al., 2015Keller P.J. Ahrens M.B. Freeman J. Light-sheet imaging for systems neuroscience.Nat. Methods. 2015; 12: 27-29Crossref PubMed Scopus (48) Google Scholar). Neuroscientists often focus on analyzing data from the cell bodies of neurons being imaged, but these signals are contaminated by those from closely passing axons and dendrites (Figures 1A and 1B ) (Harris et al., 2016Harris K.D. Quiroga R.Q. Freeman J. Smith S.L. Improving data quality in neuronal population recordings.Nat. Neurosci. 2016; 19: 1165-1174Crossref PubMed Scopus (103) Google Scholar, Peron et al., 2015Peron S.P. Freeman J. Iyer V. Guo C. Svoboda K. A Cellular Resolution Map of Barrel Cortex Activity during Tactile Behavior.Neuron. 2015; 86: 783-799Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, Chen et al., 2013Chen T.-W. Wardill T.J. Sun Y. Pulver S.R. Renninger S.L. Baohan A. Schreiter E.R. Kerr R.A. Orger M.B. Jayaraman V. et al.Ultrasensitive fluorescent proteins for imaging neuronal activity.Nature. 2013; 499: 295-300Crossref PubMed Scopus (2918) Google Scholar). Computational methods attempt to algorithmically correct somatic signals for the neuropil contribution (Pinto and Dan, 2015Pinto L. Dan Y. Cell-Type-Specific Activity in Prefrontal Cortex during Goal-Directed Behavior.Neuron. 2015; 87: 437-450Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, Andilla and Hamprecht, 2014Andilla F.D. Hamprecht F.A. Sparse Space-Time Deconvolution for Calcium Image Analysis.in: Ghahramani Z. Welling M. Cortes C. Lawrence N.D. Weinberger K.Q. Advances in Neural Information Processing Systems. 27. 2014: 64-72Google Scholar, Mukamel et al., 2009Mukamel E.A. Nimmerjahn A. Schnitzer M.J. Automated analysis of cellular signals from large-scale calcium imaging data.Neuron. 2009; 63: 747-760Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar, Pnevmatikakis et al., 2014Pnevmatikakis E.A. Gao Y. Soudry D. Pfau D. Lacefield C. Poskanzer K. Bruno R. Yuste R. Paninski L. A structured matrix factorization framework for large scale calcium imaging data analysis.arXiv. 2014; (arXiv:1409.2903)http://arxiv.org/abs/1409.2903Google Scholar, Pnevmatikakis et al., 2016Pnevmatikakis E.A. Soudry D. Gao Y. Machado T.A. Merel J. Pfau D. Reardon T. Mu Y. Lacefield C. Yang W. et al.Simultaneous Denoising, Deconvolution, and Demixing of Calcium Imaging Data.Neuron. 2016; 89: 285-299Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar). Although such algorithms are widely used in two-photon calcium imaging, one-photon calcium imaging is subject to higher neuropil contamination levels, which remains an open problem for ongoing computational research (Zhou et al., 2016Zhou P. Resendez S.L. Rodriguez-Romaguera J. Jimenez J.C. Neufeld S.Q. Giovannucci A. Friedrich J. Pnevmatikakis E.A. Stuber G.D. Hen R. et al.Efficient and accurate extraction of in vivo calcium signals from microendoscopic video data..Elife. 2016; 7: e28728Crossref Scopus (127) Google Scholar). Alternatively, genetically encoded calcium indicators can be expressed in the nucleus, which eliminates the neuropil signal (Kim et al., 2014Kim C.K. Miri A. Leung L.C. Berndt A. Mourrain P. Tank D.W. Burdine R.D. Prolonged, brain-wide expression of nuclear-localized GCaMP3 for functional circuit mapping.Front. Neural Circuits. 2014; 8: 138Crossref PubMed Scopus (14) Google Scholar, Nguyen et al., 2016Nguyen J.P. Shipley F.B. Linder A.N. Plummer G.S. Liu M. Setru S.U. Shaevitz J.W. Leifer A.M. Whole-brain calcium imaging with cellular resolution in freely behaving Caenorhabditis elegans.Proc. Natl. Acad. Sci. USA. 2016; 113: E1074-E1081Crossref PubMed Scopus (148) Google Scholar, Schrödel et al., 2013Schrödel T. Prevedel R. Aumayr K. Zimmer M. Vaziri A. Brain-wide 3D imaging of neuronal activity in Caenorhabditis elegans with sculpted light.Nat. Methods. 2013; 10: 1013-1020Crossref PubMed Scopus (182) Google Scholar, Bengtson et al., 2010Bengtson C.P. Freitag H.E. Weislogel J.M. Bading H. Nuclear calcium sensors reveal that repetition of trains of synaptic stimuli boosts nuclear calcium signaling in CA1 pyramidal neurons.Biophys. J. 2010; 99: 4066-4077Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, Vladimirov et al., 2014Vladimirov N. Mu Y. Kawashima T. Bennett D.V. Yang C.T. Looger L.L. Keller P.J. Freeman J. Ahrens M.B. Light-sheet functional imaging in fictively behaving zebrafish.Nat. Methods. 2014; 11: 883-884Crossref PubMed Scopus (150) Google Scholar); however, the requirement for calcium to enter the nucleus slows the temporal precision of such imaging, compared with classical cytosolic calcium imaging. We hypothesized that if we could localize a genetically encoded calcium indicator such as GCaMP6f (Chen et al., 2013Chen T.-W. Wardill T.J. Sun Y. Pulver S.R. Renninger S.L. Baohan A. Schreiter E.R. Kerr R.A. Orger M.B. Jayaraman V. et al.Ultrasensitive fluorescent proteins for imaging neuronal activity.Nature. 2013; 499: 295-300Crossref PubMed Scopus (2918) Google Scholar) or GCaMP7f (Dana et al., 2019Dana H. Sun Y. Mohar B. Hulse B.K. Kerlin A.M. Hasseman J.P. Tsegaye G. Tsang A. Wong A. Patel R. et al.High-performance calcium sensors for imaging activity in neuronal populations and microcompartments.Nat. Methods. 2019; 16: 649-657Crossref PubMed Scopus (199) Google Scholar) to the cytosol near the cell body, we could greatly reduce neuropil fluorescence, similar to the effect of nuclear-localized GCaMP6f, while not sacrificing speed. Although soma targeting of membrane proteins such as optogenetic actuators has been done before (Greenberg et al., 2011Greenberg K.P. Pham A. Werblin F.S. Differential targeting of optical neuromodulators to ganglion cell soma and dendrites allows dynamic control of center-surround antagonism.Neuron. 2011; 69: 713-720Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, Shemesh et al., 2017Shemesh O.A. Tanese D. Zampini V. Linghu C. Piatkevich K. Ronzitti E. Papagiakoumou E. Boyden E.S. Emiliani V. Temporally precise single-cell-resolution optogenetics.Nat. Neurosci. 2017; 20: 1796-1806Crossref PubMed Scopus (99) Google Scholar, Pégard et al., 2017Pégard N.C. Mardinly A.R. Oldenburg I.A. Sridharan S. Waller L. Adesnik H. Three-dimensional scanless holographic optogenetics with temporal focusing (3D-SHOT).Nat. Commun. 2017; 8: 1228Crossref PubMed Scopus (66) Google Scholar, Baker et al., 2016Baker C.A. Elyada Y.M. Parra A. Bolton M.M. Cellular resolution circuit mapping with temporal-focused excitation of soma-targeted channelrhodopsin.eLife. 2016; 5: e14193Crossref PubMed Scopus (69) Google Scholar, Wu et al., 2013Wu C. Ivanova E. Zhang Y. Pan Z.-H. rAAV-mediated subcellular targeting of optogenetic tools in retinal ganglion cells in vivo.PloS one. 2013; 8: e66332Crossref PubMed Scopus (54) Google Scholar, Forli et al., 2018Forli A. Vecchia D. Binini N. Succol F. Bovetti S. Moretti C. Nespoli F. Mahn M. Baker C.A. Bolton M.M. et al.Two-Photon Bidirectional Control and Imaging of Neuronal Excitability with High Spatial Resolution In Vivo.Cell Rep. 2018; 22: 3087-3098Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), this strategy has not been adapted for genetically encoded calcium indicators. We screened through natural and artificial peptides and discovered two motifs that, when fused to GCaMP, enabled it to express within 50 μm of the cell body. Kinetics were similar to those mediated by conventional GCaMP. In intact brain circuits of larval zebrafish and mice, such soma-targeted GCaMPs greatly reduced the number of neuropil contamination spikes mistakenly attributed to a given neural cell body and reduced artifactual correlations between nearby neurons. We fused various peptides to GCaMP6f and GCaMP7f and assessed their ability to target GCaMP to the cell body (Tables S1A, list of the proteins, and S7, sequences of the fragments used). These included the kainate receptor subunit KA2 (Valluru et al., 2005Valluru L. Xu J. Zhu Y. Yan S. Contractor A. Swanson G.T. Ligand binding is a critical requirement for plasma membrane expression of heteromeric kainate receptors.J. Biol. Chem. 2005; 280: 6085-6093Crossref PubMed Scopus (51) Google Scholar, Shemesh et al., 2017Shemesh O.A. Tanese D. Zampini V. Linghu C. Piatkevich K. Ronzitti E. Papagiakoumou E. Boyden E.S. Emiliani V. Temporally precise single-cell-resolution optogenetics.Nat. Neurosci. 2017; 20: 1796-1806Crossref PubMed Scopus (99) Google Scholar), the potassium channel KV2.1 (Lim et al., 2000Lim S.T. Antonucci D.E. Scannevin R.H. Trimmer J.S. A novel targeting signal for proximal clustering of the Kv2.1 K+ channel in hippocampal neurons.Neuron. 2000; 25: 385-397Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar), the sodium channels NaV1.2 and NaV1.6 (Garrido et al., 2003Garrido J.J. Giraud P. Carlier E. Fernandes F. Moussif A. Fache M.P. Debanne D. Dargent B. A targeting motif involved in sodium channel clustering at the axonal initial segment.Science. 2003; 300: 2091-2094Crossref PubMed Scopus (267) Google Scholar), the adaptor protein AnkyrinG (Zhang and Bennett, 1998Zhang X. Bennett V. Restriction of 480/270-kD ankyrin G to axon proximal segments requires multiple ankyrin G-specific domains.J. Cell Biol. 1998; 142: 1571-1581Crossref PubMed Scopus (99) Google Scholar), and the rat small conductance calcium-activated potassium channel rSK1 (Bowden et al., 2001Bowden S.E.H. Fletcher S. Loane D.J. Marrion N.V. Somatic colocalization of rat SK1 and D class (Ca(v)1.2) L-type calcium channels in rat CA1 hippocampal pyramidal neurons.J. Neurosci. 2001; 21: RC175Crossref PubMed Google Scholar). In addition, we explored de novo designed coiled-coil proteins that self-assemble into complexes, hypothesizing that their mutual binding could potentially slow their diffusion from the cell body (Moll et al., 2001Moll J.R. Ruvinov S.B. Pastan I. Vinson C. Designed heterodimerizing leucine zippers with a ranger of pIs and stabilities up to 10(-15) M.Protein Sci. 2001; 10: 649-655Crossref PubMed Scopus (104) Google Scholar, Selgrade et al., 2013Selgrade D.F. Lohmueller J.J. Lienert F. Silver P.A. Protein scaffold-activated protein trans-splicing in mammalian cells.J. Am. Chem. Soc. 2013; 135: 7713-7719Crossref PubMed Scopus (23) Google Scholar). For some proteins, earlier work analyzed cell body expression by fusing the full-length proteins to reporters; specifically, NaV1.2, NaV1.6, AnkyrinG, and rSK1 were fused to fluorescent proteins (FPs) (Garrido et al., 2003Garrido J.J. Giraud P. Carlier E. Fernandes F. Moussif A. Fache M.P. Debanne D. Dargent B. A targeting motif involved in sodium channel clustering at the axonal initial segment.Science. 2003; 300: 2091-2094Crossref PubMed Scopus (267) Google Scholar, Schäfer et al., 2010Schäfer M.K.E. Nam Y.C. Moumen A. Keglowich L. Bouché E. Küffner M. Bock H.H. Rathjen F.G. Raoul C. Frotscher M. L1 syndrome mutations impair neuronal L1 function at different levels by divergent mechanisms.Neurobiol. Dis. 2010; 40: 222-237Crossref PubMed Scopus (26) Google Scholar, Zhang and Bennett, 1998Zhang X. Bennett V. Restriction of 480/270-kD ankyrin G to axon proximal segments requires multiple ankyrin G-specific domains.J. Cell Biol. 1998; 142: 1571-1581Crossref PubMed Scopus (99) Google Scholar, Moruno Manchon et al., 2015Moruno Manchon J.F. Uzor N.E. Dabaghian Y. Furr-Stimming E.E. Finkbeiner S. Tsvetkov A.S. Cytoplasmic sphingosine-1-phosphate pathway modulates neuronal autophagy.Sci. Rep. 2015; 5: 15213Crossref PubMed Scopus (54) Google Scholar), KA2 was fused to a Myc tag (Valluru et al., 2005Valluru L. Xu J. Zhu Y. Yan S. Contractor A. Swanson G.T. Ligand binding is a critical requirement for plasma membrane expression of heteromeric kainate receptors.J. Biol. Chem. 2005; 280: 6085-6093Crossref PubMed Scopus (51) Google Scholar), and KV2.1 was fused to an HA tag (Lim et al., 2000Lim S.T. Antonucci D.E. Scannevin R.H. Trimmer J.S. A novel targeting signal for proximal clustering of the Kv2.1 K+ channel in hippocampal neurons.Neuron. 2000; 25: 385-397Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). In some cases, key fragments were found to cause soma targeting of a reporter: for NaV1.2 and NaV1.6, 326- and 27-amino acid segments within intracellular loops between transmembrane domains, termed NaV1.2(I–II) and NaV1.6(II–III), respectively, were sufficient for somatic localization (Garrido et al., 2001Garrido J.J. Fernandes F. Giraud P. Mouret I. Pasqualini E. Fache M.P. Jullien F. Dargent B. Identification of an axonal determinant in the C-terminus of the sodium channel Na(v)1.2.EMBO J. 2001; 20: 5950-5961Crossref PubMed Scopus (120) Google Scholar, Garrido et al., 2003Garrido J.J. Giraud P. Carlier E. Fernandes F. Moussif A. Fache M.P. Debanne D. Dargent B. A targeting motif involved in sodium channel clustering at the axonal initial segment.Science. 2003; 300: 2091-2094Crossref PubMed Scopus (267) Google Scholar). For KV2.1, a 65-amino acid segment within the intracellular loop between transmembrane domains IV and V (KV2.1 motif) sufficed (Wu et al., 2013Wu C. Ivanova E. Zhang Y. Pan Z.-H. rAAV-mediated subcellular targeting of optogenetic tools in retinal ganglion cells in vivo.PloS one. 2013; 8: e66332Crossref PubMed Scopus (54) Google Scholar, Lim et al., 2000Lim S.T. Antonucci D.E. Scannevin R.H. Trimmer J.S. A novel targeting signal for proximal clustering of the Kv2.1 K+ channel in hippocampal neurons.Neuron. 2000; 25: 385-397Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). For rSK1, the tail region (rSK1 tail) sufficed (Fletcher et al., 2003Fletcher S. Bowden S.E.H. Marrion N.V. False interaction of syntaxin 1A with a Ca(2+)-activated K(+) channel revealed by co-immunoprecipitation and pull-down assays: implications for identification of protein-protein interactions.Neuropharmacology. 2003; 44: 817-827Crossref PubMed Scopus (21) Google Scholar). For AnkyrinG, it was found that the spectrin-binding domain (AnkSB motif), the tail domain (AnkTail motif), the membrane-binding domain (AnkMB motif), the COOH-terminal domain (AnkCT motif), and the serine-rich domain (AnkSR motif) were targeted to the axon and the cell body of neurons (Zhang and Bennett, 1998Zhang X. Bennett V. Restriction of 480/270-kD ankyrin G to axon proximal segments requires multiple ankyrin G-specific domains.J. Cell Biol. 1998; 142: 1571-1581Crossref PubMed Scopus (99) Google Scholar). We made more than 30 fusions between GCaMP6f and the protein fragments reported earlier (Tables S1B, fusions screened, and S7, sequences). For NaV1.2, NaV1.6, KV2.1, and rSK1, we performed fusions in which the previously characterized localization fragment was attached to the C terminus of GCaMP6f. In a recent study (Shemesh et al., 2017Shemesh O.A. Tanese D. Zampini V. Linghu C. Piatkevich K. Ronzitti E. Papagiakoumou E. Boyden E.S. Emiliani V. Temporally precise single-cell-resolution optogenetics.Nat. Neurosci. 2017; 20: 1796-1806Crossref PubMed Scopus (99) Google Scholar), we fused the channelrhodopsin CoChR (Klapoetke et al., 2014Klapoetke N.C. Murata Y. Kim S.S. Pulver S.R. Birdsey-Benson A. Cho Y.K. Morimoto T.K. Chuong A.S. Carpenter E.J. Tian Z. et al.Independent optical excitation of distinct neural populations.Nat. Methods. 2014; 11: 338-346Crossref PubMed Scopus (947) Google Scholar) to the first 150 amino acids of the KA2 receptor subunit (KA2(1–150)), thereby creating a somatic CoChR. Because both N- and C-terminal fusions of KA2(1–150) with CoChR caused somatic localization, we made similar upstream and downstream fusions of this fragment with GCaMP6f. In the present study, we also found that the first 100 amino acids of KA2 (KA2(1–100)) were sufficient to introduce somatic localization of GCaMP6f; therefore, we made additional upstream and downstream fusions of KA2(1–100) with GCaMP6f. In some cases, we inserted into the construct a superfolder GFP (sfGFP) (Pédelacq et al., 2006Pédelacq J.-D. Cabantous S. Tran T. Terwilliger T.C. Waldo G.S. Engineering and characterization of a superfolder green fluorescent protein.Nat. Biotechnol. 2006; 24: 79-88Crossref PubMed Scopus (1276) Google Scholar), which contains three mutations to EGFP to enhance folding, with a mutation to abolish its fluorescence (here called nullsfGFP). For AnkyrinG fragments, we made fusions both upstream and downstream of GCaMP6f. For de novo coiled-coil proteins, we made downstream fusions only. We expressed each of these GCaMP6f fusion proteins in cultured mouse hippocampal neurons. Using wide-field fluorescence microscopy, we screened for expression level (fluorescence under baseline conditions), somatic localization, toxicity (assessed as the percentage of dead cells), and whether there were fluorescence changes (df/f0) in response to spontaneous neural activity. Five constructs did not result in obvious toxicity, exhibited somatic localization, and displayed df/f0 similar to that of GCaMP6f. These were GCaMP6f fused to the following fragments: NaV1.2(I–II) (GCaMP6f-27-NaV1.2(I–II)-ER2); nullsfGFP and KA2(1–100) (GCaMP6f-24-nullsfGFP-24-KA2(1–100)-ER2); a zero-photocurrent CoChR mutant called nullCoChR, followed by the KV2.1 motif (nullCoChR-12-GCaMP6f-KV2.1-motif); the AnkTail motif (GCaMP6f-27-AnkTail-motif-ER2) (Figure 1D); and the coiled-coil peptide set EE-RR (GCaMP6f-27-EE-RR) (Figure 1E). We next screened these somatic GCaMP6f candidates in mouse brain slices exposed to 1 mM 4-aminopyridine (4-AP, which resulted in ∼5–20 GCaMP fluorescent transients per minute, aka GCaMP spikes). We assessed df/f0, the ratio of df/f0 between the cell body and the neuropil, and the brightness. We found that GCaMP6f-24-nullsfGFP-24-KA2(1–100)-ER2 expressed in the neurites of pyramidal neurons in the cortex, in contrast to the culture data, and did not pursue this construct further. The remaining constructs had a similar df/f0 compared with GCaMP6f and a soma-to-neuropil df/f0 ratio higher than that of GCaMP6f. GCaMP6f-27-AnkTail-motif-ER2 and GCaMP6f-27-EE-RR had the highest baseline brightness (Figure 2; Table S2, statistics for Figure 2); thus, we pursued these two constructs for more detailed characterization, naming them SomaGCaMP6f1 and SomaGCaMP6f2, respectively. We co-expressed GCaMP6f, SomaGCaMP6f1, or SomaGCaMP6f2 with the red FP miRFP to serve as a cellular tracer, using cultured mouse hippocampal neurons (Figures 1C–1E). We found that fluorescence decreased at a higher rate along the neurites in SomaGCaMP6f1-expressing cells (Figures 1F and 1G) and SomaGCaMP6f2-expressing cells (Figures 1F and 1H) compared with GCaMP6f-expressing cells (Table S2, statistics for Figure 1). We also fused GCaMP7f (Dana et al., 2019Dana H. Sun Y. Mohar B. Hulse B.K. Kerlin A.M. Hasseman J.P. Tsegaye G. Tsang A. Wong A. Patel R. et al.High-performance calcium sensors for imaging activity in neuronal populations and microcompartments.Nat. Methods. 2019; 16: 649-657Crossref PubMed Scopus (199) Google Scholar) to the EE-RR sequence, which we selected because of its biorthogonality and high level of expression in vivo (Figure 2I), to yield GCaMP7f-27-EE-RR, termed SomaGCaMP7f (Figures 1I and 1J), which was also soma localized (Figures 1K and 1L). The baseline fluorescence levels of cells expressing GCaMP6f, SomaGCaMP6f1, SomaGCaMP6f2, and GCaMP6f-NLS (nuclear localization sequence) in culture were similar to each other (Figure 3A; Table S2, statistics for Figure 3), as were the baseline fluorescence levels of GCaMP7f and SomaGCaMP7f (Figure 3A). The fluorescent responses of each molecule to a single action potential (AP) (Figure 3B) were similar between targeted and untargeted GCaMPs (Figure 3C). SomaGCaMPs had SNRs (signal-to-noise ratios; defined as the magnitude of fluorescence change caused by a single AP divided by the standard deviation of the baseline fluorescence) similar to untargeted GCaMPs, whereas GCaMP6f-NLS had an SNR lower than that of GCaMP6f (Figure 3D). We found that SomaGCaMPs had rise (τon) and decay (τoff) times, for a single AP, similar to those of untargeted GCaMP but that, as expected from previous work, GCaMP6f-NLS had τon and τoff times significantly slower than those of GCaMP6f (Figures 3E and 3F). The resting potential, membrane capacitance, holding current, and membrane resistance of cultured hippocampal neurons did not differ for cells expressing conventional versus soma-targeted GCaMPs, nor did AP width, amplitude, or threshold (Figure S1; Table S5, statistics for Figure S1). In addition, we quantified the distribution of native proteins along axons and found no differences in the locations of endogenous proteins assessed (KV2.1, NaV1.2, and the calcium channel CaV2.1, as well as the scaffolding protein AnkG) between conventional versus soma-targeted GCaMP-expressing neurons (Figure S2; Table S6, statistics for Figure S2). Both SomaGCaMPs and conventional GCaMPs appeared to express in the cytosol, as opposed to on the membrane (Figure S3). We repeated some localization experiments of Figure 1 in neurons in mouse brain slices, focusing on GCaMP6f variants for concreteness. We co-expressed GCaMP variants with a red FP (mScarlet) in layer 2/3 neurons of the mouse cortex. We used mScarlet to manually trace cells and quantified fluorescence brightness for various GCaMP6f variants (Figure 4A). We normalized the GCaMP6f fluorescence in the green channel by the mScarlet fluorescence to control for the varying size and shape of neural processes and found that for SomaGCaMPs, this ratio decreased to a few percent of the ratio for GCaMP6f-expressing cells over the first 150 μm of neurite (Figure 4B; Table S3, statistics for Figure 4). Similar patterns held when we looked at GCaMP6f brightness without mScarlet normalization (Figure 4D). We next characterized whether soma targeting of GCaMP6f could reduce neuropil contamination in brain slices, choosing SomaGCaMP6f1 for this experiment. We found that the baseline brightness of the cell body of SomaGCaMP6f1-expressing neurons was about 5-fold lower than that of GCaMP6f-expressing neurons in live brain slices (Figures 2I and S4; Table S6, statistics for Figure S4). Slices expressing GCaMP6f versus SomaGCaMP6f1 had comparable densities (18 ± 7 versus 21 ± 5 cells per 106 μm3; mean ± standard error of the mean is reported unless otherwise indicated; n = 3 slices from 3 mice each). We increased the excitation light power in SomaGCaMP6f1 experiments to match the baseline brightness of GCaMP6f (Figure 4C) for the remaining experiments of Figure 4; in such a condition, the df/f0 and SNR of GCaMP spikes per single patch-reported spikes observed during 4-AP-evoked activity were similar between GCaMP6f- and SomaGCaMP6f1-expressing cells (Figures 4E and 4F). The df/f0 of the GCaMP transient driven by a burst (5–20 spikes) was significantly higher in SomaGCaMP6f1- versus GCaMP6f-expressing cells (Figure S5; Table S6, statistics for Figure S5). We then measured the number of fluorescent GCaMP-reported spikes that lacked an associated patch-reported spike in brain slices expressing GCaMP6f versus SomaGCaMP6f1, when slices were exposed to 0.1 mM 4-AP to cause asynchronous spiking (Figure 4G versus Figure 4H). Patch-reported spike rates were similar between GCaMP6f- and SomaGCaMP6f1-expressing cells (Figure 4I). GCaMP6f neurons exhibited a roughly 2:3 ratio of erroneous spikes to actual spikes, but in SomaGCaMP6f1 slices, the ratio was reduced to 1:6 (Figure 4J). GCaMP6f-expressing neurons exhibited 10.4 ± 2.2 GCaMP spikes per minute (Figure S5D), similar to the number of electrophysiology-derived APs (Figure 4I, 6.2 ± 1.3) plus the number of erroneous spikes (Figure 4J, 3.9 ± 1.4). SomaGCaMP6f1-expressing neurons exhibited 6.7 ± 3.0 GCaMP spikes per minute (Figure S5D), similar to the number of electrophysiology-derived APs (Figure 4I, 6.0 ± 2.4) plus the number of erroneous spikes (Figure 4J, 0.65 ± 0.3). Algorithms for neuropil contamination reduction for one-photon calcium imaging have been developed for neuroscience use. A popular algorithm is constrained non-negative matrix factorization (CNMF) (Pnevmatikakis et al., 2016Pnevmatikakis E.A. Soudry D. Gao Y. Machado T.A. Merel J. Pfau D. Reardon T. Mu Y. Lacefield C. Yang W. et al.Simultaneous Denoising, Deconvolution, and Demixing of Calcium Imaging Data.Neuron. 2016; 89: 285-299Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar), which enables identification of GCaMP-expressing neurons with subsequent demixing and deconvolution of their fluorescence spikes. We simulated calcium transients in mouse (Figures 5A–5C) and larval zebrafish (Figures 5D–5F) brain, to help us understand the impact of SomaGCaMP versus CNMF on live brain imaging. We simulated the ground-truth spikes in the cell bodies (Figures 5A and 5D), as well as how the data would look in isolated volumes when imaged through a lightsheet microscope (chosen because of its high spatial resolution), reported by GCaMP6f variants (Figures 5B and 5E) versus SomaGCaMP6f variants (Figures 5C and 5F). We simulated in-plane and out-of-plane artifacts of neuropil driven by the point-spread function of the microscope and then calculated the correlation between the simulated ground-truth spiking and the microscope-observed spiking that would be observed when expressing GCaMP6f versus SomaGCaMP6f variants. We found that for both mice (Figure 5G) and fish (Figure 5H), the correlation between the simulated ground-truth spiking and the microscope-observed spiking reported by SomaGCaMP variants was significantly higher than when the microscope-observed spiking was reported by GCaMP6f (Table S4, statistics for Figure 5). CNMF, in contrast, did not increase the correlation between the simulated ground-truth spiking and the microscope-observed spiking reported by either GCaMP6f or SomaGCaMP variants. Thus, CNMF may reduce correlations in calcium imaging data, but at least in simulation, some of that reduction in correlation may be reduction in actual signal. We transiently and sparsely expressed GCaMP6f, SomaGCaMP6f1, SomaGCaMP6f2, GCaMP7f, or SomaGCaMP7f, along with mCherry as a cell morphology marker, in the brains of larval zebrafish (Figures 6A and 6B ). All of these molecules expressed successfully, but we focused on comparing GCaMP6f and SomaGCaMP6f1, because the SomaGCaMP6f1 fish transgenic line was the first to be ready for experimentation. We evaluated the green-to-red ratio for SomaGCaMP6f1 and SomaGCaMP7f and found it to decrease to a few percent over the first 150 μm of neurite coming out of the cell body (Figure 6C). We imaged the tectum of the fi" @default.
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• W3037807978 date "2020-08-01" @default.
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• W3037807978 title "Precision Calcium Imaging of Dense Neural Populations via a Cell-Body-Targeted Calcium Indicator" @default.
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